The role of acyl-CoA binding proteins in selective autophagy

酰基辅酶A结合蛋白在选择性自噬中的作用

• 批准号: 8538965
• 负责人: Taras Y. Nazarko
• 金额: $ 11.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8538965
• 关键词: Acyl Coenzyme A; Address; Adipose tissue; Adult; American; Amino Acid Motifs; Arabidopsis; Autophagocytosis; Autophagosome; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Biology; Biometry; Cell Line; Cells; Co-Immunoprecipitations; Collaborations; Complex; Consultations; Coronary heart disease; Cytoplasm; Cytosol; Development; Diazepam Binding Inhibitor; Eating; Electron Microscopy; Faculty; Family; Fatty acid glycerol esters; Fluorescence Microscopy; Genes; Goals; Grant; Health; Hela Cells; Hepatocyte; Homeostasis; Homologous Gene; Human; Immunofluorescence Microscopy; In Vitro; International; Journals; Knock-out; Knowledge; Lecithin; Lipid Binding; Lipids; Liposomes; Lysosomes; Malignant neoplasm of cervix uteri; Mammalian Cell; Mammals; Manuscripts; Mediating; Membrane; Mentored Research Scientist Development Award; Mentors; Modeling; Molecular; Monitor; Mutate; Names; National Institute of Diabetes and Digestive and Kidney Diseases; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Organelles; Orthologous Gene; Pathway interactions; Phosphatidylethanolamine; Phospholipids; Physiological; Pichia; Pilot Projects; Positioning Attribute; Prevention; Process; Proteins; Public Health; Public Speaking; Publications; Rattus; Reporter; Research; Research Personnel; Risk Factors; Role; Stroke; Structure; Study models; Techniques; Teratocarcinoma; Testing; Training; Vacuole; Vesicle; Western Blotting; Work; Writing; Yarrowia lipolytica; Yeast Model System; Yeasts; career development; design; lipid metabolism; meetings; mutant; novel; novel strategies; novel therapeutic intervention; novel therapeutics; obesity treatment; peroxisome; programs; protein complex; receptor; research and development; yeast two hybrid system

DESCRIPTION (provided by applicant): Obesity is a major health problem of the 21st century. Identification of novel approaches for prevention and treatment of obesity is of great importance for public health. Obesity specifically refers to an excessive amount of adipose tissue, where lipids are stored in specialized organelles called lipid droplets (LDs). Recent studies in the autophagy field suggest that LDs can be selectively delivered to and degraded by lysosomes. This discovery has opened an exciting, new opportunity to correct lipid metabolism in humans by modulating lipophagy, the selective autophagy (or the cell's "self-eating") of LDs. I have studied selective autophagy for the past 12 years and identified several proteins required for the selective degradation of another organelle, the peroxisome. Recently, we found that acyl-CoA binding proteins (ACBPs), Acb1 and another protein we identified as Atg36, are essential for selective autophagy of peroxisomes (pexophagy) and Ape1 complexes (Cvt pathway) in the Pichia pastoris yeast. Therefore, the first goal of my project is to elucidate the molecular role o ACBPs in selective autophagy in P. pastoris. We predict that the role of ACBPs in selective autophagy is conserved from yeast to mammals. Thus, the second goal is to elucidate the physiological role of ACBPs in mammalian cells. We will characterize the role of ACBD5 and DBI, the mammalian orthologs of Atg36 and Acb1, respectively, in both pexophagy and lipophagy. The third goal is to develop the obesity model yeast, Yarrowia lipolytica, as a simple lipophagy model and study the molecular role of ACBPs in lipophagy. My long- term goals are to develop an independent research program on lipophagy, uncover its molecular mechanism and the role of ACBPs in this process. Knowledge of the proteins specifically involved in the autophagic degradation of LDs might help to design new therapeutic approaches to cure obesity in humans. To complete my transition from (1) yeast to mammalian cells, (2) pexophagy to lipophagy and (3) mentored to an independent research, I will continue to receive training in working with mammalian cell lines and in monitoring mammalian pexophagy and lipophagy. I will do it through collaboration with Dr. Till and consultation with Dr. Cuervo, experts on mammalian pexophagy and lipophagy, respectively. I will expand my knowledge on the structure and function of ACBPs through consultation with an ACBP expert, Dr. Loomis. I will also receive extensive training on lipid biology in the lab of my co-mentor, Dr. Field, an expert on cellular lipids and lipid-binding proteins. This training is essential to accomplish lipid profiing of ACBP mutants and to study interactions of ACBPs with phospholipids. To increase my knowledge on selective autophagy and LDs, I will continue to attend both autophagy and lipid biology meetings. For the training in public speaking, I will practice giving talks at the Toastmasters International. In 2012, I will give a talk on Atg36 at the Gordon Research Seminar on Autophagy (Ventura, CA). To become a successful independent investigator, I will also receive training in biostatistics, scientific management, writing and grant writing by taking classes and through personal meetings with UCSD faculties. I will write the manuscript on the role of ACBPs in selective autophagy and submit it to one of the top-ranked journals by the end of the 2nd year of the K01 award. Additionally, I will write my first R01 grant on the molecular mechanism of lipophagy and submit it to NIDDK by the end of the 3rd year. I will start searching for a tenure-track faculty position during the 4th year and accept an offer by the end of the 4-year K01 award. Having developed an independent research direction on lipophagy, I will continue to uncover its molecular mechanism and the role of ACBPs in this process in both yeast and mammalian cells. My mentor, Dr. Subramani, will closely monitor my research and career development during the K01 award. My proposal consists of the three Specific Aims. Aim 1 will address two alternative, but not mutually exclusive, roles of ACBPs in selective autophagy in P. pastoris: (1) the role in bridging cargo and the phagophore, an autophagic isolation membrane, via interaction of ACBPs with the phagophore protein, Atg8 (yeast two-hybrid and co-immunoprecipitation studies) and (2) the role in generating phagophore membrane curvature via the interaction of ACBPs with phospholipids and remodeling of the phagophore membrane (in vitro filter and liposome binding assays, lipid profiling of ACBP mutants). Aim 2 will explore the physiological role of ACBPs in selective autophagy in mammalian cells. We will study the role of ACBD5 and DBI in pexophagy using human cervical cancer, hepatocarcinoma and teratocarcinoma cells and a new pexophagy reporter, mRFP-GFP-SKL. Our pilot studies suggest that human ACBD5 is essential for pexophagy. I will also address the role of ACBD5 and DBI in lipophagy using an established rat hepatocyte cell line and lipophagy assays. Finally, Aim 3 will develop the first simple model for lipophagy using Y. lipolytica. We will knock out the Y. lipolytica ATG36 and ACB1 genes, and elucidate their roles in lipophagy using fluorescence microscopy and a novel Tgl3-GFP processing assay. We expect studies on ACBPs in yeast and mammalian cells will extend our understanding of organelle homeostasis and provide new targets for the prevention and treatment of obesity in humans.

描述（由申请人提供）：肥胖是 21 世纪的一个主要健康问题。确定预防和治疗肥胖的新方法对于公共卫生具有重要意义。肥胖特指过量的脂肪组织，其中脂质储存在称为脂滴（LD）的特殊细胞器中。自噬领域的最新研究表明，LD 可以选择性地传递至溶酶体并被溶酶体降解。这一发现为通过调节脂质自噬（LD 的选择性自噬（或细胞的“自食”））来纠正人类脂质代谢开辟了一个令人兴奋的新机会。在过去的 12 年里，我一直在研究选择性自噬，并确定了选择性降解另一种细胞器（过氧化物酶体）所需的几种蛋白质。最近，我们发现酰基辅酶A结合蛋白（ACBP）、Acb1和我们鉴定为Atg36的另一种蛋白质对于毕赤酵母中过氧化物酶体（pexophagy）和Ape1复合物（Cvt途径）的选择性自噬至关重要。因此，我的项目的首要目标是阐明 ACBP 在毕赤酵母选择性自噬中的分子作用。我们预测 ACBP 在选择性自噬中的作用从酵母到哺乳动物都是保守的。因此，第二个目标是阐明 ACBP 在哺乳动物细胞中的生理作用。我们将描述 ACBD5 和 DBI（分别是 Atg36 和 Acb1 的哺乳动物直系同源物）在 pexophagy 和 lipophagy 中的作用。第三个目标是开发肥胖模型酵母 Yarrowia lipolytica 作为简单的脂肪自噬模型，并研究 ACBP 在脂肪自噬中的分子作用。我的长期目标是开发一个关于脂肪自噬的独立研究项目，揭示其分子机制以及 ACBP 在此过程中的作用。了解专门参与 LD 自噬降解的蛋白质可能有助于设计新的治疗方法来治疗人类肥胖。为了完成从（1）酵母到哺乳动物细胞、（2）自噬到脂肪自噬以及（3）指导独立研究的转变，我将继续接受有关哺乳动物细胞系工作以及监测哺乳动物自噬和脂肪自噬的培训。我将通过与哺乳动物自噬和脂肪自噬方面的专家 Till 博士合作并咨询 Cuervo 博士来完成这项工作。我将通过咨询 ACBP 专家 Loomis 博士来扩展我对 ACBP 结构和功能的了解。我还将在我的共同导师 Field 博士的实验室接受有关脂质生物学的广泛培训，他是细胞脂质和脂质结合蛋白方面的专家。这种训练对于完成 ACBP 突变体的脂质分析以及研究 ACBP 与磷脂的相互作用至关重要。为了增加我对选择性自噬和 LD 的了解，我将继续参加自噬和脂质生物学会议。对于公开演讲的训练，我将在国际演讲会练习演讲。 2012 年，我将在戈登自噬研究研讨会（加利福尼亚州文图拉）上发表关于 Atg36 的演讲。为了成为一名成功的独立研究者，我还将通过上课以及与加州大学圣地亚哥分校教员的个人会面来接受生物统计学、科学管理、写作和资助写作方面的培训。我将撰写关于 ACBP 在选择性自噬中的作用的手稿，并在 K01 奖第二年年底之前将其提交给顶级期刊之一。此外，我将撰写我的第一个关于脂肪吞噬分子机制的 R01 资助，并在第三年年底之前将其提交给 NIDDK。我将在第四年开始寻找终身教授职位，并在 4 年 K01 奖结束时接受聘书。在开发了关于脂肪吞噬的独立研究方向后，我将继续揭示其分子机制以及 ACBP 在酵母和哺乳动物细胞中的这一过程中的作用。我的导师 Subramani 博士将在 K01 奖期间密切关注我的研究和职业发展。我的建议包括三个具体目标。目标 1 将解决 ACBP 在毕赤酵母选择性自噬中的两种替代但不相互排斥的作用：(1) 通过 ACBP 与噬菌体蛋白 Atg8 相互作用，桥接货物和噬菌体（一种自噬隔离膜）的作用（酵母双杂交和免疫共沉淀研究）和 (2) 生成噬菌体膜的作用 通过 ACBP 与磷脂的相互作用和吞噬细胞膜的重塑来改变曲率（体外过滤器和脂质体结合测定、ACBP 突变体的脂质分析）。目标 2 将探讨 ACBP 在哺乳动物细胞选择性自噬中的生理作用。我们将使用人宫颈癌、肝癌和畸胎癌细胞以及新的 pexophagy 报告基因 mRFP-GFP-SKL 研究 ACBD5 和 DBI 在 pexophagy 中的作用。我们的初步研究表明，人类 ACBD5 对于 pexophagy 至关重要。我还将使用已建立的大鼠肝细胞系和脂肪吞噬测定来阐述 ACBD5 和 DBI 在脂肪吞噬中的作用。最后，Aim 3 将使用解脂耶氏酵母开发第一个简单的脂肪自噬模型。我们将敲除解脂耶氏酵母 ATG36 和 ACB1 基因，并使用荧光显微镜和新型 Tgl3-GFP 处理测定法阐明它们在脂肪自噬中的作用。我们期望对酵母和哺乳动物细胞中 ACBP 的研究将扩展我们对细胞器稳态的理解，并为人类肥胖的预防和治疗提供新的靶标。